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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 26, 2005 as doi:10.1096/fj.05-3881fje. |
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* Département de Biologie Cellulaire de l’Institut Cochin, The Institut National de la Recherche Médicale (INSERM U567), The Centre National de la Recherche Scientifique (UMR 8104), and the Université René Descartes, Paris France; and
INSERM U683, Paris, France
1Correspondence: Département de Biologie Cellulaire, Institut Cochin, Hôpital Cochin, Pavillon G. Roussy 5 Etage, 27 rue du Faubourg St. Jacques, Paris 75014, France. E-mail: perianin{at}cochin.inserm.fr
SPECIFIC AIMS
Chemoattractant receptors play a key role in cell trafficking and pathological processes (inflammation, metastasis). Chemoattractant-induced inflammatory cell responses are mediated through the activation of various intracellular signaling effectors. Among these, phosphatidylcholine-specific phospholipase D (PLD) is a major source of second messengers (phosphatidic acid, diglycerides, phosphocholine) that regulates various physiological processes including phagocyte defense activities, as well as neoplastic transformation. The biochemical regulation of PLD activity involves protein phosphorylation. So far, a role of mitogen-activated (MAP) kinases in chemoattractant-mediated PLD phosphorylation and activation remains unknown.
In this study, we have used pharmacological, biochemical, and molecular biology approaches in HL-60 and HEK 293T cells to investigate 1) the contribution of p44/42 MAP kinases (ERK1/2) to formyl-peptide-induced cellular PLD activity; 2) the respective contribution of the PLD1 and PLD2 isoform; 3) the phosphorylation of PLD2 by ERK in vivo and in vitro; and 4) the functional contribution of the previously undescribed ERK1/2/PLD coupling to formyl-peptide-mediated production of oxidant in HL-60 cells.
PRINCIPAL FINDINGS
1. The PLD activity mediated by the formyl-peptide chemoattractant receptor in HL-60 cells or HEK 293T cells is strongly dependent on ERK1/2 activation
Stimulation of differentiated HL-60 cells or HEK cells expressing formyl-peptide receptor by the chemotactic peptide f-Met-Leu-Phe (fMLP) induced a rapid and potent stimulation of PLD activity (Fig. 1
A), which was completely inhibited when ERK1/2 activation was prevented with the MEK1/2 antagonist U0126 (10 µM) (Fig. 1B-D
). Conversely, the expression of a constitutively activated MEK1 mutant in HEK 293T cells potentiated (P<0.05) fMLP-induced PLD activity. U0126 also inhibited PLD activity mediated by the chemokine receptors, CCR5 and CXCR4, expressed in HEK cells, whereas it did not alter basal or PLD activity induced by the direct protein kinase C (PKC) activator, phorbol myristate acetate (PMA).
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2. Contribution of PLD1 and PLD2 to fMLP-mediated PLD activity in HEK 293T cells and ERK1/2-mediated in vivo phosphorylation of PLD2
Two distinct isoforms of PLD, PLD1 and PLD2 are expressed in most cell types. Transient expression of catalytically inactive PLD1 or PLD2 mutants in HEK 293T cells or treatment of HL-60 cells with PLD antisense oligonucleotides showed that PLD2 was essential in fMLP-mediated cellular PLD activity. PLD2 coimmunoprecipitated with ERK1/2 in resting cells and became phosphorylated on MAP kinase consensus sites (S/T-P) in fMLP-stimulated HEK cells, as determined by Western blot analysis (Fig. 2
). This PLD2 phosphorylation was inhibited by U0126, indicating it was dependent on ERK1/2 activation. In cell-free systems, a constitutively activated ERK1/2 directly phosphorylated the immunopurified PLD2 that generates only two phosphopeptides after tryptic digestion and 2-dimensional peptide mapping. In isolated cell membranes from resting HEK 293T cells, purified ERK1/2 was also able to stimulate a rapid and transient PLD activity in the presence ATP, independent of PKC.
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3. The ERK/PLD2 pathway regulates fMLP-induced respiratory burst of HL-60 cells
Stimulation of PLD activity in neutrophils by fMLP regulates the production of reactive oxygen species (respiratory burst, RB), which serve as microbicidal agents and contribute to physiopathological processes. The inhibition of the ERK/PLD pathway by U0126 was associated with a strong but partial reduction of fMLP-mediated RB (
65%). This reduction linearly correlated with the inhibition of PLD activity (R=0.924, P<0.01). In addition, the fMLP-mediated PLD activity and RB was not altered by the selective PKC antagonist GF 109203X, which, however, strongly inhibited PMA-induced PLD activity and RB, as expected. Thus, the ERK1/2-PLD pathway may contribute predominantly to the fMLP-induced RB of HL-60 cells; a predominant implication of the PLD2 isoform in RB was shown by antisense oligonucleotides.
CONCLUSIONS AND SIGNIFICANCE
This study provides new insights to the current model of PLD regulation by chemoattractants (Fig. 3
). The PLD activity mediated by the fMLP receptor or the chemokine receptor, CCR5 and CXCR4, was regulated by ERK1/2. The PLD2 isoform was the main effector implicated and was phosphorylated through a ERK1/2-dependent manner. Pharmacological modulation of PLD2 phosphorylation in vivo and in vitro studies prompt us to suggest that PLD2 stimulation by ERK may occur through its phosphorylation on very few sites, which remains to be identified. This previously undescribed ERK/PLD2 coupling contributes to the production of oxidant production in fMLP-stimulated neutrophilic HL-60 cells, suggesting a role in host defense mechanisms.
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Given the importance of ERK and PLD in various short- and long-term cellular physiological activities (cell differentiation, proliferation) our data may provide insights not only into defense mechanisms but also into physiological regulatory processes. Identification of molecular links between ERK and PLD2 may provide pharmacological targets for the development of mimetics endowed with protective properties against hyperactivation of PLD and related cellular dysfunctions.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3881fje;
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